Receptor membranes and ionophore gating

ABSTRACT

The present invention provides a membrane the conductivity of which is dependent on the presence or absence of an analyte. The membrane comprises a closely packed array of self-assembling amphiphilic molecules and two ionophore components. A receptor molecule reactive with the analyte is provided on one of the ionophore components. The binding of the analyte to the receptor molecule causes a change in the relationship between the ionophore components such that the flow of ion across the membrane is prevented or allowed. The ionophore components are preferably selected from the group consisting of amphotericin B, gramicidin A monomers and combinations thereof, with gramicidin A monomers being particularly preferred. The present invention also provides a membrane including receptors directed against the Fc region of antibodies. These receptors are preferably derived from polyclonal antibodies. These membranes provide a &#34;generic&#34; surface which will bind antibodies in a manner such that the antigen binding regions of the antibody are not hindered. The present invention further provides a device adapted for implantation in a mammalian body, the device being characterized in that it is coated with a membrane comprising a closely packed array of self-assembling amphiphilic molecules and receptor molecule. The receptor molecules being such that the attachment of specific cells to the membrane is enhanced or avoided. It is particularly preferred that the receptor molecules are directed against fibronectin, vitronectin, endothelial cells, or epithelial cells. It is further preferred that the membrane coating the device also includes a plurality of ion channels such as gramicidin.

This is a Continuation of application Ser. No. 08/449,962, filed 25 May1995, now abandoned, which is a continuation in part of Ser. No.07/721,431, filed Jul. 3, 1991 now U.S. Pat. No. 5,443,950.

The present invention relates to a membrane bilayer in which each layerhas incorporated therein ionophores and in which the conductance of themembrane is dependant on the presence or absence of an analyte. Thepresent invention further relates to membranes including receptorsdirected against the Fc region of antibodies. In addition, the presentinvention also relates to devices adapted for implantation in amammalian body, the surface of the device being coated with a membranewhich includes receptors.

It is known that amphiphilic molecules may be caused to aggregate insolution to form two dimensional membrane arrays in a variety ofmorphologies such as monolayers, black lipid membranes, vesicles andliposomes. It is also known that such amphiphilic molecules may beformed with cross linkable moieties. Under appropriate stimulus, such asUV radiation or ionizing radiation, the cross-linkable moieties can becaused to polymerize after the amphiphilic molecules have been caused toassume a suitable ordered two dimensional array. It is also known thatsuitable receptor molecules may be included in ordered arrays ofamphiphilic molecules.

The selectivity and flux of ions through membranes can depend on thenumber, size and detailed chemistry of the pores or channels theypossess. It is through these pores or channels that the permeatingsolubilized molecules pass across the membrane, It is also known thatmembranes may incorporate a class of molecules, called ionophores whichfacilitate the transport of ions across these membranes.

In co-pending application No. WO 89/01159 it is disclosed that suitablymodified receptor molecules may be caused to co-disperse withamphiphilic molecules and produce membranes with altered surface bindingproperties, which are useful in the production of biosensor receptorsurfaces of high binding ability and high binding specificity. It isalso disclosed in this co-pending application that ion channels such aspolypeptide ionophores may be co-dispersed with amphiphilic molecules,thereby forming membranes with altered properties in relation to thepermeability of ions. This application also discloses various methods ofgating these ion channels such that in response to the binding of ananalyte the conductivity of the membrane is altered. The disclosure ofapplication No. WO 89/01159 is incorporated herein by way of reference.

When membranes formed from these various components are maintained at atemperature above a critical temperature, Tc, variously known as thetransition temperature, chain melting temperature or phase transitiontemperature, the ion channels, and receptor molecules present in themembrane are able to diffuse laterally within the two dimensional planeof the membrane.

Immunoglobins possess characteristics such as high specificity, highaffinity, variety and stability, which make them ideal for use asanalytical reagents. Antigen-antibody interactions form the basis ofsensitive diagnostic techniques such as radioimmunoassay (RIA) andenzyme-linked immunosorbant assay (ELISA). Although these techniques aresensitive and widely used, commercial antibody-based diagnostic kitswhich include disposable single-use devices are semi-quantitative,time-consuming and cannot be calibrated which limits their analyticalpotential. The optimum immuno-based assay system should be fast,reliable, specific, sensitive and quantitative.

ELISA's and RIA's commonly immobilize the antibody via non-covalentassociation of antibodies with glass or plastic surfaces. This typicallyblocks many antibody binding sites, reducing activity and preventingprecise quantitation of the number and affinity of the remaining sites.More recently, orientation of antibody binding sites has been achievedby specific attachment of the antibody or antibody fragments, F(ab)₂ andFab, to a surface via groups remote from the antigen binding region. Thebinding between the antibody and antigen can be detected by a variety ofmethods some of which use electrical measurements. However, thisprocedure requires that for each new analyte to be detected, thespecific antibody raised against the analyte must be affinity purifiedfrom a polyclonal mixture so that further reduction to the F(ab)₂ or Fabfragments can be carried out.

The present invention involves the orientation of the antigen-bindingportion of the chosen monoclonal or polyclonal antibody such that theantigen-binding site remains free and unhindered. The monoclonal statusis not a prerequisite for the antigen detection technique, and furtherreduction to F(ab)₂ or Fab fragments is not required. The antibodyraised against the analyte to be detected (A) is bound to acylatedantibody or antibody fragments (B) incorporated into a self-assembledmonolayer or bilayer membrane composed of amphiphilic molecules.

It is also believed that membranes including receptor molecules may beused to coat devices for implantation, and that such coating wouldrender the surface biocompatible.

Biocompatible surfaces are needed for any invasive prosthetic device.Depending on the type of device and the location within the body, theprosthetic device needs to have a surface which will encourage theadhesion of certain cells while repelling other types of cells, Forexample, adsorption of plasma proteins such as fibrinogen or attachmentof cells such as erythrocytes can lead to thrombosis, and adsorption ofcells to surfaces which lead to tissue build-up, are both undesirableconsequences of interfacing tissue with a foreign surface. However, atthe point of entry of an invasive prosthetic device into the body,cell-surface adhesion can act as a seal against the spread of bacteriaand hence infection along the device and into the body.

A strategy to promote the beneficial adhesion between the surface of theinvasive device and desired cells, whilst at the same time preventingthe attachment of undesirable cells and plasma proteins, is to encouragethe adhesion to the surface of the invasive device of specific cellssuch as epithelial or endothelial cells via their cell surfaceattachment proteins such as fibronectin and vitronectin.

In a first aspect the present invention consists in a membrane in whichthe conductance of the membrane is dependent on the presence or absenceof an analyte, the membrane comprising a closely packed array ofamphiphilic molecules and a plurality of ionophores comprising a firstand second half membrane spanning monomer, at least the second halfmembrane spanning monomer being capable of lateral diffusion within themembrane, a first receptor molecule provided on at least the second halfmembrane spanning monomers, said first receptor molecule being reactivewith the analyte or a portion thereof, the binding of the analyte to thefirst receptor molecule causing a change in the relationship between thefirst half membrane spanning monomer and the second half membranespanning monomer such that the flow of ions across the membrane via theionophores is allowed or prevented.

In a preferred embodiment of the present invention the membranecomprises a first and a second layer, the first half membrane spanningmonomers being provided in the first layer and the second half membranespanning monomers being provided in the second layer.

The half membrane spanning monomer can be any of such molecules known inthe art, however, it is presently preferred that the monomers areselected from the group consisting of gramicidin A monomers,amphotericin B, and combinations thereof. It is most preferable that themonomers are gramicidin A monomers.

In a preferred embodiment of a first aspect of the present invention thefirst half membrane spanning monomers in the first layer are preventedfrom lateral diffusion within said first layer and the second halfmembrane spanning monomers in the second layer are capable of lateraldiffusion within said second layer.

The first half membrane spanning monomer in the first layer may beprevented from diffusing laterally using any of a number of knowntechniques, however, it is presently preferred that the monomer and theamphiphilic molecules each include or are decorated with at least onemoiety cross-linked with at least one corresponding moiety on another ofthese molecules. Under appropriate stimulus, such as UV radiation orionizing radiation, the cross-linkable moieties can be caused topolymerize thereby resulting in the membrane being cross-linked in onelayer.

The first half membrane spanning monomers may also be prevented fromdiffusing laterally by selecting lipids for the first layer of themembrane which are crystalline at room temperature. This eliminateslateral diffusion in the first layer.

In a further preferred embodiment of the present invention the firsthalf membrane spanning monomer in the first layer is prevented fromdiffusing laterally by fixing the first layer and the monomers thereinto a solid support. This may be achieved by providing groups on theamphiphilic molecules in the first layer and on the monomers thereinwhich are reactive with the solid support or with corresponding groupsprovided thereon.

In a further preferred embodiment of this aspect of the presentinvention the membrane includes a plurality of second receptor moleculeshaving receptor sites. It is also preferred that the second receptormolecules are prevented from diffusing laterally within the membrane.

In the situation where the membrane is a bilayer it is preferred thatthe second receptor molecule is provided in the second layer such thatIts receptor sites project outwardly from the surface of the secondlayer remote from the first layer.

As used herein the term "receptor molecule" is used in its widestcontext. The receptor molecule may be any chemical entity capable ofbinding to the desired analyte. The receptor molecule is any compound orcomposition capable of recognize another molecule. Natural receptorsinclude antibodies, enzymes, lectins, dyes, chelating agents and thelike. For example the receptor for an antigen is an antibody, while thereceptor for an antibody is either an anti-antibody or, preferably, theantigen recognised by that particular antibody. In addition, thereceptor for an ion such as calcium would be the chelating agent EDTA.

The first and second receptor molecules may be the same or different andare preferably selected from the group consisting of polyclonal ormonoclonal antibodies, fragments thereof including at least one Fabfragment, antigens, lectins, haptens, chelating agents and dyes and aremost preferably antibodies or fragments thereof.

It is also preferred that first receptor molecule, and optionally thesecond receptor molecule, has two receptor sites for the analyte.

The second receptor molecule is preferably an antibody or fragmentthereof including at least one Fab fragment or antibody. When thereceptor molecule is an Fab fragment or antigen this may be conjugatedwith a supporting entity such as is described in application WO89/01159. The second receptor molecule may be prevented from diffusinglaterally within the second layer by any of a number of known means suchas cross-linking of the receptor molecule to amphiphilic moleculeswithin the second layer. In the situation where the receptor molecule isconjugated with a supporting entity it is possible to prevent lateraldiffusion of the receptor moiety by having the supporting entity extendthrough both the second and first layers.

When the membrane of the present invention is used in a biosensor it ispreferred that the membrane is attached to a solid surface. This may beachieved by providing groups reactive with the solid surface on theamphiphilic molecules in the membrane. Preferred solid surfaces includehydrogel, ceramics, oxides, silicon, polymers and transition metals.Preferred transition metals are gold, platinum and palladium. Theattachment of the membrane to a solid surface may be achieved bynon-covalent attraction or by covalent reactions. For example, vinylgroups on a solid substrate could be copolymerized with avinyl-terminated lipid; a sulphur-terminated lipid could be adhered to ametal (e.g. gold or palladium) substrate; or condensation or additionreactions could be used to anchor the lipid. Modification of the solidsubstrate, if necessary, can be achieved using any of the knowntechniques such as silylation or silica surfaces. In the situation wherethe membrane is a bilayer, the first layer is attached to the solidsurface.

Preferably the second layer is selected to have a phase transitiontemperature such that cooling of the membrane below this temperatureinduces phase separation in the second layer thereby releasing anyanalyte bound to either the first and/or second receptor molecules. Thisshould provide a convenient method of "resetting" the device following atest.

In preferred embodiments of this aspect of the present invention thefirst receptor is attached to the ionophore via a linker group. Thislinker group typically comprises a spacer group and one or more reactivegroups, the spacer group being attached to ionophore and the reactivegroup providing attachment to the receptor molecule. The spacer groupcan consist of hydrocarbons, oligomers of ethylene glycol,oligo-peptides etc. and is of a length such that the ionophore is ableto conduct ions when the receptor molecule is coupled. The reactivegroups can consist of N-hydroxysuccinimide esters or other commonactivated esters for covalent coupling to amine groups on proteins,hydrazine derivatives for coupling onto oxidized sugar residues;maleiimide derivatives for covalent attachment to thiol groups; biotin;streptavidin or antibodies.

The linker group may consist of a number of reactive groups, forexample, in one preferred embodiment of the present invention the linkergroup comprises a spacer group which is attached to biotin which in turnis attached to streptavidin. With this particular linker group thereceptor molecule to be bound would be a biotinylated antibody, thebiotin on the antibody binding to the streptavidin.

In a further preferred embodiment of the present invention the terminalreactive group on the linker is an antibody or antibody fragmentdirected against the Fc portion of an antibody. With such a terminalreactive group the linker will bind to the Fc region of an antibodywhich will be the receptor molecule directed against the analyte.

In a further preferred embodiment of the present invention the secondreceptor molecule is attached to the membrane using similar linkergroups.

In a preferred embodiment of the present invention the first receptormolecules on separate second half membrane spanning monomers bind todifferent sites on the analyte, such that binding of the first receptorsto the analyte results in the prevention of the flow of ions across themembrane.

In a further preferred embodiment of the present invention the firstreceptor molecules on separate second half membrane spanning monomersare bound to different sites on the analyte or an analog thereof, suchthat the flow of ions across the membrane is prevented, the addition ofanalyte effecting competitive binding with the first receptor moleculesresulting in the flow of ions across the membrane.

In yet a further preferred embodiment of the present invention at leasta proportion of the amphiphilic molecules are membrane spanningamphiphiles, the membrane spanning amphiphiles being archeobacteriallipids or tail to tail chemically linked bilayer amphiphiles. In theembodiment where the membrane exists as a mono-layer, it is preferredthat all the amphiphilic molecules are membrane spanning amphiphiles.

As is stated above one of the preferred terminal reactive groups on thelinker molecule is streptavidin. This would typically be bound to thespacer group by way of a biotin reactive group. When using such amultivalent reactive group it is essential that the linker groups arenot able to cross-link as this would result in a change in therelationship between the ionophores in the first layer and theionophores in the second layer in the absence of any analyte.Accordingly, when a molecule such as streptavidin is to be used as theterminal reactive group it is essential that the ability of thestreptavidin to cross-link with the biotin provided on other linkergroups is prevented. This may be done by ensuring that the biotinbinding site of the streptavidin adjacent to the biotin binding site bywhich the streptavidin is bound to the linker group is occupied bybiotin. This can be achieved by pre-incubating the streptavidin with anappropriate amount of biotin.

In the absence of competing influences the ionophores in each of thefirst and second layers will, on average, align themselves to produce anintact channel which allows the passage of ions through the membrane.When the ionophores in the second layer diffuse out of alignment withthe ionophores in the first layer, the channel will be broken and ionswill not pass through the membrane. With this arrangement the diffusionof the ionophores may, when incorporated into a suitable membrane, beused to detect as little as a single molecule of analyte. The attachmentof a single molecule of analyte may cause an intact ion channel to beformed or broken either allowing or stopping the flow of ions across themembrane. After a brief time this change in passage of ions across themembrane may be detected as the signal for the binding of the analyte toa receptor. The measurement of current flow across membranes due to asingle ionophore is known and typically yields a current of 4 pA perchannel.

In a second aspect the present invention consists in a membrane in whichthe conductance of the membrane is dependent on the presence or absenceof an analyte, the membrane comprising a closely packed array ofamphiphilic molecules and a plurality of membrane spanning helicalpeptide aggregate ionophores comprising a plurality of membrane spanninghelical peptide monomers each provided with a receptor molecule reactivewith the analyte, binding of the analyte to the receptor moleculecausing disruption of the membrane spanning helical peptide aggregate.

In a preferred embodiment of this aspect of the present invention themembrane spanning helical peptide monomers are alamethicin monomers.

In a further preferred embodiment of this aspect of the presentinvention the receptor molecules are selected from the group consistingof polyclonal or monoclonal antibodies, antibody fragments including atleast one Fab fragment, antigens, lectins, haptens, chelating agents anddyes. However, at present it is preferred that the receptor moleculesare antibody fragments including at least one Fab fragment andpreferably an Fab fragment.

The membrane may exist as either as a bilayer or monolayer and in thesituation where the membrane is a monolayer, it is preferred that theamphiphilic molecules are membrane spanning amphiphiles such asarcheobacterial lipids or tail to tail chemically linked bilayeramphiphiles.

When the membrane of this aspect of the present invention is used as abiosensor it is preferred that the membrane is attached to a solidsurface. This may be achieved by providing groups reactive with thesolid surface on the amphiphilic molecules in the membrane. Preferredsolid surfaces include hydrogel, ceramics, oxides, silicon, polymers andtransition metals. Preferred transition metals are gold, platinum andpalladium. The attachment of the membrane to a solid surface may beachieved by non-covalent traction or by covalent reactions.

In further preferred embodiments of this aspect of the present inventionthe receptor molecules are attached to the membrane spanning helicalpeptide monomers via a linker group. This linker group is as previouslydescribed in the first aspect of the present invention.

As is known, alamethicin ionophores consist of aggregates of alamethicinmonomers which associate to form conducting ionophores. It is believedthat the aggregation/association of alamethicin monomers in a membranewould be prevented if these monomers are provided with a receptor groupwhich is subsequently bound to an analyte.

In a third aspect the present invention consists in a membrane for usein detecting the presence of an analyte, the membrane comprising aclosely packed array of self-assembling amphiphilic molecules wherein atleast a proportion of the self-assembling amphiphilic molecules comprisea receptor molecule conjugated with a supporting entity, the receptormolecule having a receptor site and being reactive with the Fc region ofan antibody, the receptor molecule being selected from the groupconsisting of the Fc binding domain of an Fc receptor, an antibody,F(ab)₂ and Fab fragments; the supporting entity being selected from thegroup consisting of a lipid head group, a hydrocarbon chain(s), across-linkable molecule and a membrane protein; the supporting entitybeing conjugated with the receptor molecule at an end remote from thereceptor site, and in which antibody molecules reactive with the analyteare bound to the receptor molecule by their Fc region.

As used herein the term "Fc receptor" is defined as cell membranereceptors reactive against the Fc portion of immunoglobulin.

In a preferred embodiment of this aspect of the present invention thereceptor molecules are preferably derived from polyclonal antibodies. Itis also presently preferred that the receptor molecules are covalentlyattached to the supporting entity via their amino acid side chains orcarbohydrate moieties.

In a further preferred embodiment of the present invention, the receptormolecules conjugated to the supporting entity are able to diffuselaterally through the membrane bilayer or monolayer.

In a further preferred embodiment of the present invention, the antibodyreactive with the analyte is a monoclonal antibody. It is also presentlypreferred that the antibody reactive with the analyte may consist of twoor more different monoclonal antibodies directed against differentepitopes present on the same analyte.

As would be readily appreciated by a person skilled in the art, by usingreceptor molecules directed against the Fc portion of species-specificantibodies, for example anti-mouse Fc antibodies or F(ab)₂ of Fabfragments thereof, the membrane of the present invention can be usedwith any mouse antibody.

The membrane of the present invention is generally first prepared fromantibodies raised against the Fc portion of species-specific antibodies,eg. anti-mouse Fc antibodies. In the situation where antibody fragmentsare used these anti Fc antibody fragments (B) are preferably covalentlyattached to the supporting entity via their terminal amino acid sidechain or carbohydrate moieties and incorporated into the self-assembledamphiphilic monolayer or bilayer. The antibody (A) raised against thedesired analyte to be detected may be of monoclonal or polyclonalnature. The antibody (A) is then bound to the self-assembled monolayeror bilayer via its Fc region by the anti Fc antibody fragments (B) whichare incorporated into the monolayer or bilayer.

Where the receptor molecule is the Fc binding domain of an Fc receptor,it is preferred that the Fc receptor is selected from the class ofreceptors known as Fc δ RI or Fc δ RII or Fc δ RIII. It is particularlypreferred that the Fc receptor is reactive against the Fc portion ofIgG.

As stated above only the Fc binding domain of the Fc receptor is used,optionally together with the transmembrane domain. Whilst this portioncould be generated by isolating the whole receptor molecule, it isbelieved to be preferable to produce the extracellular Fc binding domainor the extracellular Fc binding domain together with the transmembranedomain using genetic engineering techniques. To achieve this clonedgenes encoding the Fc receptor would be modified to produce only therelevant domain(s). This will, of course, involve deletion of a portionof the gene sequence.

It is also envisaged that further modifications of the gene sequence maybe made for both Fc receptors and antibodies. These include, forexample, for the extracellular domain of the Fc receptor, or theantibodies addition of one or more amino acids including a cysteineresidue to provide a sulphydryl group which may be required for chemicalcross-linking of the Fc receptor or antibody binding domain to themembrane molecules. This would be achieved by site directed mutagenesis.

It is also envisaged that modifications may be made to the gene sequenceencoding the Fc binding domain to increase the affinity of the receptorfor bound antibody and/or to increase the range of IgG molecules boundby Fc δ RI, Fc δ RII and/or Fc δ RIII. Modifications may also be made tothe gone sequence of the antibody binding domain to increase bindingaffinity.

The lipid matrix of the membrane ensures the incorporation of theantibody and the correct orientation of each antibody molecule. Thisself-assembled monolayer or bilayer may be formed by any of the knownmethods such as the Langmuir-Blodgett technique, liposomes or BLM.

Preferably detection of antigen-binding interactions is carried outusing transduction measurements. It is preferred that the membrane ofthe present invention is attached to a solid support such as a metal,polymer or hydrogel. The detection mechanism may be achieved by choosinga conducting solid support which can detect the antigen-antibody bindingevent. The detection of the binding event will depend on the preferredphase separation of the antibody-antigen complexes from the amphiphilicmolecules in the membrane on the surface of the conducting solidsupport, thus bare conducting solid support is exposed to an aqueousenvironment thereby changing the transduction measurement. The phaseseparation of the antibody attached to freely diffusing antibodyfragments incorporated into the membrane depends on the binding of thedesired antigen by at least two different antibodies raised against thesame analyte. The binding of two or more antibodies induces clusteringof the antibodies which alters the phase of the monolayer or bilayerrendering it "leaky" to the aqueous environment. This "leakiness"changes the transduction property of the conducting solid support whichis no longer insulated from the aqueous environment by the membrane.

The density of antibodies present in the membrane can be varied byvarying the ratio of amphiphile to antibody-supporting entity conjugate.Increased stability of the membrane of the present invention may beachieved by placing the membrane on a solid support. Appropriatetreatment of the chosen solid support enables covalent linkage of thehydrocarbon chains of the amphiphile and supporting entity to the solidsupport. The choice of amphiphile used in the membrane may be such that,upon binding of the antigen, the monolayer or bilayer phase of the solidsupport is disrupted rendering the surface "leaky", as is known in theart. Such a device can be designed to have an "all or nothing" responseto a chosen minimum quantity of antigen present in the testing solution.

In summary, this aspect of the present invention provides a membranewhich can be prepared incorporating any antibody as a receptor foranalyte detection, with minimum preparation of the antibody, Themembrane of the present invention has the following advantages overexisting devices that use antibody molecules:

1. The correct orientation of the antibody binding site is achieved forevery antibody molecule by attaching it to an integral component of theself-assembled amphiphilic monolayer or bilayer film.

2. Density of receptor molecules in the membrane can be controlled andhence optimized for the most sensitive detection of the desired analyte.

3. The membrane of the present invention prepared with anti-Fcspecies-specific receptor molecules enables the use of any antibodyraised in the selected species to be used in the membrane withoutfurther preparation of the antibody into monoclonal fractions or F(ab)₂or Fab fragments.

4. Electrical measurements provide quicker results of the binding assayby using a conducting solid support which detects changes uponantigen-binding and subsequent monolayer or bilayer phase disruptionbrought about by the aggregation of two or more antibodies bound to thesame analyte.

In a fourth aspect the present invention consists in a device adaptedfor implantation in a mammalian body, the device being coated with amembrane comprising a closely packed array of self-asembling amphiphilicmolecules in which at least a proportion of the self-assemblingamphiphilic molecules comprise a receptor molecule conjugated with asupporting entity, the receptor molecule having a receptor site, thereceptor molecule being selected from the group consisting of antibodiesand antibody fragments; the supporting entity being selected from thegroup consisting of a lipid head group, a hydrocarbon chain(s), across-linkable molecule and a membrane protein; the supporting entitybeing conjugated with the receptor molecule at an end remote from thereceptor site and in a manner such that the receptor site is at orprojects from a surface of the membrane remote from the device, thereceptor molecule being such that either the attachment of specificcells to the membrane is enhanced or avoided.

In a preferred embodiment of this aspect of the present invention themembrane also includes a plurality of ion channels. It is preferred thatthe ion channel are peptides which form a β helix, and are mostpreferably gramicidin.

In a further preferred embodiment of the second aspect of the presentinvention the receptor molecules are F(ab)₂ or Fab fragments. It is alsopreferred that the receptor molecules are directed against fibronectin,vitronectin, endothelial cells, or epithelial cells, and most preferablyfibronectin.

In a further preferred embodiment of the present invention, the membraneis attached to the device by providing groups on the membrane reactivewith the surface of the device or with groups provided thereon.

In yet a further preferred embodiment of the present invention, thereceptor molecules are either F(ab)₂ or Fab fragments which are linkedto the supporting entity via their terminal sulfhydryl group.

In another preferred embodiment of the present invention, theamphiphilic molecules and the ion channels and/or receptor moleculesupporting entity conjugate are provided with cross-linkable moieties ina manner such that each cross-linkable moiety is cross-linked to thecross-linkable moiety on another molecule.

At present it is preferred that the biocompatibility of the device isenhanced by using amphiphilic molecules naturally occurring in themammalian species into which the device is to be implanted, orderivatives of these amphiphiles or synthetic amphiphiles, provided witha thiol group for attachment to metal surfaces such as palladium,titanium, platinum, silver or gold. Use of such amphiphiles tends todecrease the degree of host-device rejection. It is also preferred thatthe antibodies or antibody fragments are also derived from the mammalianspecies into which the device is to be implanted, as this will alsoresult in a decreased level of host-device rejection. Alternatively theantibodies for antibody fragments are modified to reduce rejection e.g.humanization.

It is believed that attachment of epithelial or endothelial cells can befacilitated by the coating of the implantable device with theself-assembled monolayer or bilayer of polymerized or non-polymerizedamphiphiles which incorporate orientated acylated antibodies or antibodyfragments. The use of antibodies or antibody fragments raised againstcell surface attachment proteins such as fibronectin or vitronectinshould result in the coated implantable device binding epithelial orendothelial cells, thereby facilitating adhesion. Such a device coatedwith epithelial or endothelial cells will present a non-foreign surfaceto the body, preventing potential platelet adhesion which leads tothrombosis or tissue build-up. The cell-surface binding can also act asa seal to prevent infection spreading along a catheter inserted into thebody. Anti-fibronectin antibodies and other antibodies againstepithelial or endothelial cells are known in the art and tend to bespecies-specific.

As stated above, it is preferred that the self-assembled monolayer orbilayer is comprised of amphiphiles based on or derived from naturallyoccurring lipid molecules. Specific covalent linkage of the antibody orantibody fragments to the amphiphile ensures that each antibody bindingsite is correctly orientated such that all sites are accessible forbinding to the antigen, and binding density can therefore be controlled.

A further advantage of the present invention is that the provision ofion channels in the membrane enables the passage of ions through themembrane to the device. Amphiphiles such as lipids provide abiocompatible matrix for the coating of the implantable device which isrendered permeable to charge transfer by the incorporation of ionchannels. A number or ion channels may be used, however, at present theion channel gramicidin, and in particular, gramicidin A and analoguesthereof are preferred.

The incorporation of an ion channel such as gramicidin A may also renderthe surface of the implantable device antibacterial due to thebactericidal activity of gramicidin.

The presence of anti-epithelial or anti-endothelial antibodies in theself-assembled monolayer or bilayer consisting of amphiphiles and ionchannels can prevent platelet adhesion or tissue build-up on theimplanted device while at the same time still being permeable to chargetransfer processes. Therefore, such a membrane can be used inconjunction with implantable devices which contain electrodes such aspacemaker leads and other devices which rely on charge transfer fordetection or application while needing to present a non-thromboticsurface free from tissue build-up.

As will be appreciated by those skilled in the art, the conductance ofthe membrane of the first aspect of the present invention is dependenton the presence of the analyte due to the gating of the ionophores. Thisgating occurs due to displacement of the ionophores in one layerrelative to the ionophores in the other layer following binding of theanalyte to the receptor molecules. Three displacement gating mechanismsare possible and these have been designated "local disruption gating","extended displacement gating" and "vertical disruption gating".

In order that the nature of each of these gating mechanisms may be moreclearly understood, preferred embodiments of the first aspect of thepresent invention will now be described with reference to theaccompanying drawings, in which:

FIG. 1 shows a schematic representation of a membrane of the firstaspect of the present invention in which the binding of the analyteresults in a decrease in conductivity of the membrane due to localdisruption gating;

FIG. 2 shows a schematic representation of a membrane of the firstaspect of the present invention in which the presence of an analyteresults in a decrease in the conductivity of the membrane due toextended displacement gating;

FIG. 3 shows a schematic representation of a membrane of the firstaspect of the present invention in which the presence of an analyteresults in a decrease in conductivity of the membrane due to verticaldisruption gating;

FIG. 4 shows a schematic representation of a membrane of the firstaspect of the present invention in which the presence of the analyteresults in an increase in the conductivity of the membrane due toextended displacement gating.

FIG. 5 shows the reaction scheme used to produce modified gramicidin;

FIG. 6 shows the results of impedance measurements of the membrane ofthe first aspect of the present invention;

FIGS. 7 to 9 show the results of experiments demonstrating the gating ofthe membrane of the first aspect of the present invention;

FIG. 10 shows linker lipid A;

FIG. 11 shows linker gramicidin B;

FIG. 12 shows membrane spanning lipid XXB;

FIG. 13 shows Ga(XXXB)₂ ;

FIG. 14 shows comparison of response to ferritin challenge for membranescontaining anti ferritin Fab' or anti theophylline Fab' fragments;

FIG. 15 shows ferritin response;

FIG. 16 shows detection of ferritin at two different concentrations;

FIG. 17 shows anti-theophylline antibody titration; and

FIG. 18 shows thephylline titration.

As can be seen in FIG. 1 the membrane 10 consists of a first layer 11and second layer 12 each comprising an array of amphiphilic molecules13. Provided in first layer 11 are ionophores 14 and in second layer 12ionophores 15. Attached to an end of ionophores 15 via linker group 26are receptor molecules 16. In the absence of analyte (the situationshown in FIG. 1a) intact channels are formed due to the alignment ofionophores 14 and 15 through which ions may flow across the membrane.

As is shown in FIG. 1b, upon the addition of analyte 17, the analyte 17is bound to receptor molecules 16. This results in a cross-linking whichcauses a disturbance of alignment of ionophores 14 and 15, therebypreventing the flow of ions across the membrane by the ionophores 14 and15.

As can be seen in FIG. 2 the membrane 10 consists of a first layer 11and a second layer 12 composed of amphiphilic molecules 13. Provided infirst layer 11 are ionophores 14 which are prevented from lateraldiffusion within the first layer 11 due to cross-linking shown generallyas 25. Ionophores 15 are provided in second layer 12. Attached to an endof ionophore 15 via linker group 26 is a first receptor 18 and includedwithin second layer 12 is a second receptor 19 which is also preventedfrom diffusing laterally. In the absence of analyte (the situation shownin FIG. 2a) intact channels are formed due to the alignment ofionophores 13 and 14 through which ions may flow across the membrane.

Upon the addition of analyte 20 these intact channels are broken. As canbe seen in FIG. 2b the analyte 20 binds to first receptor 18 and secondreceptor 19. As second receptor 19 is prevented from diffusing laterallywithin second layer 12, the binding of first receptor 18 to analyte 20which is bound to second receptor 19 causes the movement of ionophore 15out of alignment with ionophore 14. This is referred to "extendeddisplacement gating".

As can be seen in FIG. 3 the membrane 10 consists of a first layer 11and second layer 12 composed of amphiphilic molecules 13. Provided infirst layer 11 are ionophores 14 and in second layer 12 ionophores 15.Attached to an end of ionophore 15 via linker group 26 is a receptormolecule 21. In the absence of analyte intact channels are formed due tothe alignment of ionophores 14 and 15.

Upon the addition of analyte 22 ionophores 15 and the second layer 12are pulled away from ionophores 14 and first layer 11. This productionof a space between the first layer 11 and second layer 12 results inchannels no longer capable of allowing the passage of ions across themembrane. This form of gating is referred to as vertical displacementgating.

An alternate arrangement involving extended displacement gating is shownin FIG. 4. As can be seen in FIG. 4a the membrane 10 consists of a firstlayer 11 and second layer 12 comprising amphiphilic molecules 13.Provided in the first layer 11 are ionophores 14 which are preventedfrom lateral diffusion within the first layer 11 due to cross-linkingshown generally as 25. Ionophores 15 are provided in second layer 12.Attached to an end of ionophore 15 via linker group 26 is a firstreceptor 22 and included within second layer 12 is a second receptor 23.In the absence of analyte (the situation shown in FIG. 4a) ionophores 14and 15 are out of alignment due to the first receptor 22 being bound tosecond receptor 23. In this case the second receptor 23 is the analyteto be detected or an analogue thereof.

As is shown in FIG. 4b, upon addition of analyte 24, by competitivebinding, the first receptor 22 is released from second receptor 23 andionophore 15 moves back into alignment with ionophore 14 therebycreating an intact channel which enables ions to flow across themembrane via ionophores 14 and 15.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following examples:

EXAMPLE 1 Linker-Gramicidin

    ______________________________________     ##STR1##    ______________________________________    gramicidin       spacer      reactive    ethanolamine     group       group    terminus    ______________________________________

spacer group can consist of hydrocarbon, oligomers of ethylene glycol,oligopeptides etc. of such a length that gramicidin is able to conductions when the receptor molecule is coupled.

reactive groups can consist of N-hydroxysuccinimide esters or othercommon activated ester for covalent coupling to amine groups onproteins, hydrazine derivatives for coupling onto oxidised sugarresidues, or maleiimide derivatives for covalent attachment to thiolgroups, biotin, streptavidin or antibodies.

Synthesis of a Modified Gramicidin for Protein Attachment

1. Compound 1 (see scheme 1)

Succinic anhydride (2 g) and benzyl alcohol (2.2 g) were dissolved inpyridine (10 ml) and heated at 45° for 18 h. The cooled mixture waspoured onto hydrochloric acid (1M, 200 ml) and extracted withdichloromethane (3×50 ml). The combined CH₂ Cl₂ extracts were dried (Na₂SO₄) and evaporated to give compound 1 as a white solid (2 g).

2. Compound 2

Compound 1 (2 g) was stirred with 80 thionylchloride (10 ml) for 3 h. atroom temperature. Excess thionylchloride was distilled and the residuewas treated with tetraethylene glycol (25 ml) and pyridine (20 ml) andstirred for 24 hours. The mixture was poured onto hydrochloric acid (1M,300 ml) and extracted with CH₂ Cl₂ (3×50 ml). The combined CH₂ Cl₂extracts were dried (Na₂ SO₄) and evaporated. The residue was filteredthrough a small plug of silica gel using ethyl acetate eluent to givethe product as a pale yellow oil (1.2 g).

3. Compound 3

Compound 2 (0.5 g) and succinic anhydride (0.2 g) were mixed in pyridine(2 ml) and stirred for 24 h. The mixture was poured onto hydrochloricacid (1M, 100 ml) and extracted with dichloromethane (3×30 ml). Thecombined CH₂ Cl₂ extracts were dried (Na₂ SO₄) and evaporated to givecompound 3 as a pale yellow oil (0.5 g).

4. Compound 4

Gramicidin (0.112 g), compound 3 (0.307 g), dicyclohexyldinimide (0.13g), and a catalytic amount of 4-(N,N-dimethylamino)-pyridine were mixedin dry dioxane (10 ml) and stirred for 24 h. Excess dioxane was removedunder reduced pressure and the residue was chromatographed on silica gel(dichloromethane/methanol/water/triethylamine 400:42:4:1 eluent) toyield the product as a white solid (0.53 g).

5. Compound 5

Compound 4 (0.02 g) was dissolved in ethanol and 10% palladium oncharcoal was added. The mixture was then hydrogenated under hydrogen at1 atm for 2 h. The mixture was filtered and the residue waschromatographed on silica gel(dichloromethane/methanol/water/triethylamine, 400:42:4:1 eluent) toyield the product as a white solid (0.19 g).

6. Compound 6

Compound (5) (0.019 g ) was dissolved in dichloromethane (3 ml) anddicyclohexyl carbodiimide (0.01 g) and N-hydroxysuccinimide (0.006 g)was added. The mixture was stirred for 24 h. and excess solvent wasevaporated. The residue was taken up in ethanol and precipitated withwater to give Compound 6 as a white solid (0.15 g).

Compound 7

Biotinylated Gramicidin A

A mixture of gramicidin A (49 mg, 27 μmol), N-BOC-glycine (48.5 mg, 277μmol), dicyclohexylcarbodiimide (28.5 mg, 138 μmol) and4-(N,N-dimethylamino)-pyridine (6.5 mg, 53 μmol) in dry, distilleddichloromethane (12 ml) was refluxed for 25 min. then allowed to cool toroom temperature over 20 min. and evaporated to dryness under reducedpressure. The residue was chromatographed on a silica gel column elutedwith dichloromethane/methanol/water/acetic acid (400:40:4:1) to afford amajor U.V.-active fraction containing O-(N-BOC-glycyl)-gramicidin A (70mg); R_(f) (CH₂ Cl₂ /MEOH/H₂ O/ACOH): 0.29.

O-Glycylgramicidin A

O-(N-BOC-glycyl)-gramicidin A (35 mg) was dissolved in redistilledtrifluoroacetic acid (2 ml) under nitrogen and the solution was swirledfor 5 min then evaporated to dryness. The residue was triturated withbenzene (4 ml) then evaporated to dryness. The product was thenchromatographed on a silica column eluted withdichloromethane/methanol/water/triethylamine (400:40:4:1) to a major,polar fraction of O-Glycylgramicidin A (33 mg).

O-(Biotinyl-ε- aminocaproyl-glycyl) Gramicidin A

To a mixture of O-Glycylgramicidin A (16 mg) and biotinyl-ε-aminocaproicacid N-hydroxy succinimide ester (3.5 mg) in dichloromethane/methanol(2:1, 1.5 ml) was added triethylamine (1 μl) and the mixture was stirredfor 28 h then evaporated to dryness. The residue was chromatographed ona silica gel column eluted with dichloromethane/methanol/water(200:30:3) to afford O-(Biotinyl-ε-aminocaproyl-glycyl)-gramcidin A (9mg).

Attachment of Antibody to Conductive Gramicidin Channels viaStreptavidin-Biotin Complex

In order to use streptavidin as a linking element for attachingreceptors to gramicidin ion channels, while still maintaining theconductivity of the channel it is clearly a prerequisite that thestreptavidin molecule attached to the gramicidin-bound biotin have theadjacent biotin-binding site occupied to prevent crosslinking of thechannel. Futhermore, a biotin-binding site on the opposite side of thestreptavidin molecule must be maintained for attachment of thebiotinylated receptor of choice. Such a configuration may be achieved ina variety of ways. One such protocol is as follows:

1. A black lipid membrane was formed from a solution of 50 mg/mlglycerol monooleate in N-decane with biotinylated gramicidin added to aconcentration of 12.5 μM. The impedance of the BLM is shown in FIG. 6 asline 30.

2. 10 μl of a preformed 1:1 complex of biotin/streptavidin was added tothe BLM resulting in an increase in impedance as shown by line 31 inFIG. 6. A significant residual conductance remained, due to conductingbiotinylated gramicidin channels.

3. 10 μl of anti-Fc antibody was added to the black lipid membrane. Thebinding of the anti-Fc antibody was evidenced by a concommittantdecrease in membrane impedance shown as line 32 in FIG. 6.

4. 25 microliters of anti-HCG antibody was added to the black lipidmembrane. The binding of the anti-HCG antibody to the anti-Fc antibodywas evidenced by an increase in impedance shown as lines 33 and 34 inFIG. 6. This increase in impedance observed following the binding of theanti-HCG antibody is evidence of the gating of the gramicidin ionchannels in the black lipid membrane.

EXAMPLE 2 N-Dansyl-dimyristoylphosphatidyl Ethanolamine

A mixture of dimyristoylphosphatidyl ethanolamine (65 mg, 0.102 mmol),dansyl chloride (37.5 mg) and triethylamine (15 μl) inchloroform/methanol (3:1, 4 ml) was stirred at room temperature for 24h. then evaporated to dryness. The residue was dissolved indichloromethane (30 ml) and washed with aqueous potassium bicarbonatesolution (2.5% w/v, 20 ml). The organic phase was separated and theaqueous phase was extracted with dichloromethane (2×10 ml). The combinedorganic phases were dried (Na₂ SO₄), filtered and evaporated to dryness.The residue was chromatographed on a silica column eluted withdichloromethane/methanol (9:1) to afford a yellow-fluorescent product(38 mg); R_(f) (CH₂ Cl₂ /MEOH, 4:1)0.41.

EXAMPLE 3 Linker LipidsN-4-(4-Maleimidophenyl)-butyryl-dimyristoylphosphatidyl Ethanolamine

To a solution of dimyristoylphosphatidyl ethanolamine (64 mg) andtriethylamine 14 μl) in chloroform/methanol (4:1, 5 ml) was added solid4-(4-maleimidophenyl)-butyric acid N-hydroxysuccinimide ester (48 mg)and the mixture was stirred at room temperature for 2 h. The mixture wasevaporated to dryness then dissolved in chloroform (30 ml), washed twicewith aqueous sodium chloride solution (1%, 20 ml), dried (Na₂ SO₄),filtered and evaporated to dryness. The residue was chromatographed on asilica column eluted successively with chloroform, chloroform/methanol(95:5), chloroform/methanol (90:10) and chloroform/methanol (80:20) toafford the title compound (53 mg).

As would be appreciated by one skilled in the art, a range of other suchlinker lipids with varying reactive and spacer groups for attachment ofreceptors may be readily prepared (J. Connor, et al (1985) Pharmacol.Ther. 28, 341-365).

EXAMPLE 4

A generic surface for antigen binding can be produced by linkingspecies-specific IgG anti-Fc antibody, antibody fragments or the Fcbinding domain of an Fc receptor to a monolayer or biolayer ofself-assembly amphiphilic molecules, orientated such that the anti-Fcmolecules binding sites were available for binding antibody molecules.Linking the anti-Fc molecule can be carried out using a variety oflinkers attached to either membrane proteins such as gramicidin or tolipids (as illustrated by Examples 1 and 3). The biolayer ofself-assembling amphiphilic molecules can be produced by liposomes, BLMor on a supported substrate.

This Example uses polyclonal anti-mouse IgG anti-Fc antibody as theanti-Fc molecule and unilamellar liposomes (small unilamillar vesicles)prepared from lipid amphiphilic molecules.

Briefly, small unilamellar vesicles were prepared and fractionated usingsonication and ultracentrifugation (C. Huang (1969) Biochemistry 8,344-350; Y. Barenholz, D. Gibbes, B. J. Litman, J. Goll, T. E. Thompson,F. D. Carlson (1977) Biochemistry 16, 2806-10; J. Surrkuusk, B. R.Lentz, Y. Barenholz, R. L. Bittonen, T. E. Thompson (1976) Biochemistry,15, 1393-1401; C. F. Schmidt, D. Lichtenberg, T. E. Thompson (1981)Biochemistry, 20, 4792-97).

Chromatographically pure egg yolk lecithin and cholesterol weredissolved in chloroform in a one-to-one ratio. The fluorescent lipidmarker, Dansyl-PE, prepared as shown in Examples 2, was added to themixture in a 1% mole ratio. Linker-gramicidin or linker lipid, preparedas shown in Examples 1 and 3 were added in a 1% mole ratio.Specifically, compound 6 in Example 1, the N-hydroxy-siccinimidederivative of gramicidin, and compound 7 in Example 1, the biotinderivative of gramicidin, were the linker-gramicidin molecules used inseparate experiments. The solvents were removed by vaccuum, and thelipids lyophilized from cyclohexane:methanol (95:5). The mixture washydrated in 100 mM phosphate buffered saline, pH 7.9, and vortexed.Lipid dispersions were sonicated using a Branson sonifier (B-12) fittedwith a cup horn sonifer, over ice for up to 30 min (3 min or 2 mincooling cycle). Small unilamellar vesicles were fractionated byultracentrifugation at 180,000 g using a Beckman Ti 75 rotor at 10° C.for 90 min. Region III containing unilamellar vesicles was removed (Y.Barenholz, D. Gibbes, B. J. Litman, J. Goll, T. E. Thompson and F. D.Carlson, (1977) Biochemistry 16, 2806-10). Approximately 10% of theoriginal material i.e. 1-5 μmoles total lipid, formed the Region IIIvesicles, as determined by the phospholipid concentration (G. R.Bartlett (1959) J. Biol. Chem. 234, 466-468) and dansyl-PE fluorescence,measured at 525 nm. Incorporation of GA or derivatives was determined byfurther fractionation of the vesicles on Sepharose CL4B and measuringGA's absorption at 280 nm of fractions containing the fluorescentdansyl-PE. The vesicle preparations were used immediately for theantibody conjugation procedure.

Anti-Fc antibodies were coupled to the linker-containing residues by thefollowing method. Anti-mouse IgG polyclonal anti-Fc antibody and I-125labelled anti-Fc antibody were added to the vesicles in 100 mM phosphatebuffered saline, pH 7.9, at a concentration of 1-5 mg/ml. (If biotin wasthe reactive group of the linker on either gramicidin or lipid,streptavidin was added to the antibody in a 1:1 mole ratio, incubated at37° C., 30', before adding to the vesicles.) The mixture was incubatedat 20° C. for 12 hours and the anti-Fc antibodies linked to the vesicleswas fractionated from unbound anti-Fc antibody by chromatography onSepharose CL-4B. Anti-Fc antibody bound to vesicles was determined fromthe specific activities of I-125 labelled antibody and the fluorescentmeasurements of the Dansyl PE in the vesicles. The fractions containinganti-Fc antibody covalently attached to the vesicles were active whenassayed by radioimmunoassay for antigen-binding activity, using mousemonoclonal IgG antibody.

SURFACE TREATMENT EXAMPLE 5

A non-cytotoxic surface can be prepared by adsorbing amphiphilicmolecules via a thiol moiety to a metal-coated surface, such astitaniam, palladium, platinum, gold or silver. The molecules may benaturally occuring lipids or their derivatives, such asphosphatidylcholines with terminal sulfhydryls (L. C. Coyl et al, 1989,Chemistry of Materials 1, 606-611) or synthetic thiol-bearing compoundssuch as alkanes (E. B. Troughton, 1988, Langmuir, 4, 365) or alkanederivatives bearing hydrophilic head-groups such polyethylene oxide.

In this example the alkane derivatives dodecane-thiol, polyethyleneoxide-thiol and dodecane polyethylene oxide-thiols were synthesized,absorbed from high-purity distilled ethanol to metal surfaces and testedfor cytotoxicity.

Palladium-coated glass slides were prepared by sputtering palladiumunder vaccuum onto clean slides, and immediately transferred todistilled ethanol solutions of thiol lipids, such as11-mecapto-3,6,9-trioxundecane-1-ol and1-(1,2-dimyristoylglyceryl)-2-(11-mercapto-3,6,9-trioxaundecane-1-yl)-succinate,dodecane thiol. Resistance measurements of the thiol-lipid coated glassslides were carried out before the cytotoxicity tests were performed.

Cytotoxicity tests investigated the changes in cell count and cellmorphology after 24 hr direct contact with the thiol-lipid coated glassslides, using bare glass and bare palladium-sputtered glass slides ascontrols. Sheep endothelial cells and mouse fibroblast cells, grown on10% foetal calf serum were added to treated-styrene cell culture dishescontaining the thiol-lipid coated glass slides. Each culture dishcontained 30,000 cells/cm².

The slides were incubated at 36.5° C. for 24 hrs in anatmosphere-controlled oven. No evidence of cell death was shown byeither viability staining or cell count. Both types of cells adherred tothe thiol-lipid coated glass slides as successfully as their adhesion tothe cell culture dish itself.

EXAMPLE 6

Antibodies raised against cell adhesion proteins such as vitronectin orfibronectin can be attached to the non-cytotoxic thiol-lipid coatedglass slides as in Example 5 via amino acid side chains such as Arg,Lys, Asp, Glu or Cys using a cross-linkable molecule on the lipid thatreacts with amino, carboxyl or sulfhydryl groups such as in Examples 1and 3. Hence provide a non-cytotoxic surface which can bind endothelialor epithelial cells. Addition of gramicidin provides a surface withcharge transfer capacity.

EXAMPLE 7 Membrane Gating

Apparatus

The black lipid membrane (BLM) apparatus consisted of two 10 cc chambersseparated by a septum containing a 0.5 mm diameter hole. The chamberscontained the BLM bathing solution and the hole in the teflon supportedthe BLM. One chamber was fitted with a glass window for the objective ofa ×10 microscope. The two chambers were fabricated of polyacrylamide(Perspex) and the septum was made from PTFE (Teflon). The chambers wereheld in place by teflon insulated stainless steel bolts. Gaskets madefrom reinforced medical grade silicon rubber were used between theperspex and teflon components. The membrane electrical impedance wasmeasured using silver/silver chloride electrodes together with a ListLM-EPC7 patch clamp amplifier and a computer controlled signalgenerator. The excitation was a sine wave swept in frequency from 0.1 Hzto 100 Hz. The voltage was set at 20 mV and the membrane currentamplification was set at 0.5 mV/pA with the bandwidth limited to 1 kHzby a 6th order bandpass filter.

The apparatus was cleaned using distilled ethanol and distilleddeionised water. Detergent should not be used for cleaning and alltraces of detergent were removed. Traces of ethanol should be removed bypumping the components in a vacuum chamber. A 12.5 μM solution ofbiotynylated gramicidin in n-decane was prepared and 100 mg/ml ofglycerol monoleate was added to the solution. A silicon rubber tube wasfitted onto the end of a 50 microliter syringe, which was filled withabout 10 microliters of the solution and used to wipe a film of lipidacross the hole in the convex face of the septum. This film formed a BLMwithin a few minutes.

Titration Test of Lateral Segregation Gate

This series of measurements was designed to demonstrate theavidin-biotynylated gramicidin gate and also to demonstrate the gatingmechanism. Streptavidin has four binding sites for biotin and thepurpose of the titration study was to determine if more than one ofthese sites was required to disrupt the biontylated gramicidin channel.Measurements were made with a series of solutions of streptavidin inwhich none, one, two, three and four biotin binding sites were blockedwith biotin.

First a BLM was formed from a solution of glycerol monoleate (50 mg/ml)and biotynylated gramicidin (12.5 μM) in n-decane which results in anionic conductance of about 250 megohms. The BLM was bathed in a 0.1Msaline solution. Ten microliters, of mole ratios 4, 3.8, 3.6 and 3.2:1biotin to streptavidin was successively added to the saline solution onone side only of the BLM. No increase in BLM impedance was observed.However, when streptavidin with no attached biotin was added to thesolution on the other side the impedance increased from about 250megohms to about 12,000 megohms. Similarly when a single addition ofmole ratios 3.2:1 was added to a freshly made BLM the impedanceincreased from 250 megohms to 8,000 megohms as before. FIG. 7 shows thebasic gate effect in which streptavidin is added to an ionicallyconducting BLM. In FIG. 7, line 40 shows the impedance of the BLMwithout streptavidin added and line 41 shows the impedance followingaddition of streptavidin. FIG. 8 shows the titration in which the 4:1(line 43), 3.8:1 (line 44) and 3.6:1 (line 45) biotin:streptavidin wereadded. Line 42 shows the impedance of the BLM containing biotinylatedgramicidin which results in an ionic conductance of about 250 megohms.It should be noted that the impedance instead of increasing actuallydecreases. FIG. 9 shows the effect of adding 3.2:1 biotin:streptavidinto a fresh BLM containing biotinylated gramicidin by itself. In FIG. 9the impedance of the membrane incorporating biotinylated gramicidin isshown as line 47, the effect of addition of streptavidin is shown as 48and the effect of the 3.2:1 biotin:streptavidin is shown as line 49.

In each of these Figures the impedance spectra have logarithmicallyscaled axes with impedance on the ordinate and frequency on theabscissa. The impedance ranges from 10 megohms to 100 megohms with theline through 3.0 representing 100 megohms. The frequency ranges from 1millihertz to 100 hertz with the line through 0.0 representing 1 hertz.Most of the impedance spectra consists of two distinct components, a 45°line at the high frequency end of the spectrum which is the capacitivecomponent of the membrane and a horizontal line which represents itsresistive component. At low impedances the resistive component cancompletely dominate the capacitive component. At high impedance changesin the capacitive component indicate changes in the morphology of themembrane while changes in resistance indicate changes in the ionchannels through the membrane.

FIG. 7 shows a BLM formed from a solution of glycerol monooleate (50mg/ml) and biotinylated gramicidin (12.5 μm) in n-decane which resultsin an ionic conductance of about 250 megohms line 40. Line 41 shows thatadding 10 microliters of 1 mg/ml streptavidin solution to the solutionon either side of the membrane, increases the impedance to about 12,000megohms.

In FIG. 8, line 42 shows a BLM containing biotinylated gramicidin whichresults in an ionic conductance of about 250 megohms. Line 43 shows thatadding streptavidin in which all the biotins sites have been blockedwith biotin, does not bind the biotinylated gramicidin and makes nodifference to the conductivity. Similarly lines 44 and 45 show thatwhere the number of biotin binding sites on the streptavidin are lessthan two there is no increase in impedance i.e. no disruption of thegramicidin has occurred, however, the reduction in impedancedemonstrates that binding has occurred. When unbiotinylated streptavidinwas subsequently added to the solution on the same side of the BLM nofurther change in the impedance occurred giving a further indiction thatbinding had occurred without gating and that gating had to be due to across-linking rather than a single binding. All the above additions wereto one side of the membrane only. When streptavidin was added to theother side of the BLM the conductance increased to about 12,000 megohmsdemonstrating that the ion channel gating mechanism was not effected bybinding of streptavidin to the biotinylated gramicidin, i.e. competitivebinding of the streptavidin was the only factor inhibiting the gagingeffect.

In FIG. 9 lines 46 shows a BLM containing a biotinylated gramicidinwhich results in an ionic conductance of about 250 megohms. Line 47shows the effect of adding biotinylated streptavidin prepared as a 3.2:1biotin:streptavidin. This shows that a significant number of doublebinding sites are available with a ratio of 3.2:1. Line 47 shows theeffect of subsequently added unbiotinylated streptavidin. The increasein impedance shown in line 47 over line 46 indicates that a significantnumber of streptavidin molecules were completely biotinylated with aratio of 3.2:1 biotin:streptavidin. This is as would be expected.

FIG. 6 shows the impedance spectra corresponding to preparation of abiosensing membrane and the change in spectra associated with thesensing of anti-HCG antibodies.

Streptavidin consists of four biotin-binding units which are arrangedwith the biotin-binding sites located in closely spaced pairs atopposite poles of the molecule. The effect of streptavidin on theconductance of a bilayer containing gramicidin with covalently attachedbiotin is herein shown to be dependent on whether one or both adjacentbiotin-binding sites directed toward the bilayer are available to thegramicidin-linked biotins. A streptavidin biotin-binding site may berendered unavailable by occupying it with a molecule of free biotin i.e.biotin not covalently attached to another species. It is expected thatthe addition of biotin to streptavidin will afford a binomialdistribution of streptavidin species with 0, 1, 2, 3 or 4 bound biotinsaccording to the ratio of biotin to streptavidin. This distribution isset out in Table 1.

                  TABLE 1    ______________________________________    Ratio Biotin:              % Streptavidin Species with    Streptavidin              0         1      2     3   4 bound biotins    ______________________________________    0.1       100       0       0     0   0    1.1       31        42     21     5   1    2:1       6         25     38    25   6    3:1       1         5      21    42  31    3.2:1     1         3      15    41  40    3.6:1     1         1       5    29  66    3.8:1     1         1       1    17  81    ______________________________________

It is also evident that streptavidin with no bound biotins will containonly species with two adjacent binding sites and streptavidin with threebound biotins will contain only species with one biotin binding sitewhile those with one or two biotins will contain mixtures of one and twoadjacent biotin binding sites. Thus samples of streptavidin containingin excess of single binding sties over adjacent available binding sitescan be prepared by adding large ratios of biotin to streptavidin.

EXAMPLE 8 Ferritin Gating in Presence or Absence of Tethered Receptor

Assembly of the Biosensor Membrane

Onto a clean glass of plastic slide, an adhesion layer of chromium (50angstrom) followed by a gold layer (200-2000 angstroms) is evaporated.The freshly evaporated gold coated electrode is taken and immediatelyassembled into a teflon slide assembly holder such that an electrodesurface is defined by a circular teflon well pressed onto the goldelectrode. The teflon well forms a tight, water impermeable seal at theelectrode perimeter. 5 μl of an ethanolic solution of glycerylmonophytanyl ether (10 mM), linker lipid A, FIG. 10 (1 mM), thedisulfide of mercaptoacetic acid (0.8 mM), linker gramicidin B, FIG. 11(0.1 μM), membrane spanning lipid XXB, FIG. 12 (1 μM), is added to eachwell. (Synthesis protocols for linker lipid A, linker gramicidin B, andmembrane spanning lipid XXB are set out in WO 92/17788 published on Oct.15, 1992 (corresponding to U.S. Ser. No. 08/119,116, filed Sep. 21,1993, now U.S. Pat. No. 5,401,378, issued Mar. 28, 1995; Atty. Dkt.47-53) and WO 94/07593, published on Apr. 14, 1994 (corresponding toU.S. Ser. No. 08/406,853, filed May 17, 1995, Atty. Dkt. 47-75). Thegold coated electrode is left immersed in the solution for 5 minutes,and rinsed with ethanol before the second layer is added.

Formation of the second layer of the bilayer membrane is carried out byaddition of 5 μl of a solution containing 28 mM of glyceryl monophytanylether and 0.28 μM Ga(XXXB)₂ (FIG. 13). The well assembly was then rinsed2 times with phosphate buffered saline (PBS) resulting in the formationof the second lipid layer of the bilayer sensing membrane. The wellassembly holds approximately 200 μl of PBS. Into this 200 μl of PBS inthe well is placed a counter electrode a connection is made between thiscounter electrode and impedance bridge measuring apparatus. To completethe electrical circuit, the other connection is made between the goldelectrode and the impedance bridge. Using the impedance bridge theconduction of the membrane may then be determined.

Ferritin Response

The ferritin detection is carried out using anti-ferritin antibody Fab'fragments attached to both the gramicidin channel and the tetheredmembrane spanning lipid C via a streptavidin-biotin linker.

Streptavidin (5 μl 0.5 mg/ml PBS) is added to the membrane and left toincubate 10-60 min, before rinsing out with PBS. Anti-ferritin Fab'fragments, biotinylated via the free thiol groups using standardmethods, is added to the membranes (5 μl 0.05 mg/ml PBS), incubated10-60 min, then rinsed out with PBS. Ferritin in PBS is added to thebiosensor and the change in impedance response is recorded against time.

FIGS. 14 and 15 show the ferritin response in the presence or absence ofanti-ferritin Fab' fragments. The control membrane in FIG. 14 wasprepared without Fab' fragments and shows no response to ferritinaddition. The control membrane in FIG. 15 was prepared withanti-theophylline Fab' fragments (which show no specificity orselectivity toward ferritin) and hence did not respond to the additionof ferritin. FIG. 16 demonstrates the detection of ferritin at twodifferent concentrations--21×10⁻⁹ M and 120×10⁻¹² M.

EXAMPLE 9 Theophylline Detection

Bilayer Assembly

Onto a clean glass to plastic slide, an adhesion layer of chromium (50Angstroms) followed by a gold layer (200-2000 Angstroms) is evaporated.The freshly evaporated gold coated electrode is assembled onto a teflonslide assembly holder such that an electrode surface is defined by acircular teflon well (diameter=4 mm, volume=200 μl) pressed onto thegold electrode. The teflon well forms a tight, water impermeable seal atthe electrode perimeter. Onto the electrode is dispensed a 2 μl aliquotof an ethanolic solution comprising linker lipid A (10 mM), thedisulfide of mercaptoacetic acid (0.8 mM), linker gramicidin B (0.1 μM),and membrane spanning lipid XXB (1 μM), followed immediately by additionof ethanol (50 μl). The gold coated electrode is incubated with thesolution for 30 minutes. The ethanolic solution is then removed from thewell and the well is refilled with ethanol (50 μl). This process isrepeated twice and the final ethanol wash is removed, leaving the wellempty. This procedure forms the first layer of the bilayer sensormembrane and may be stored in ethanol, glycerol, ethylene glycol orother alcohol for several months. Formation of the second layer of thebilayer membrane is carried out by addition of 2 μl of an ethanolicsolution of glyceryl monophytanyl ether (10 mM) andgramicidin-theophylline conjugate (0.1 μM), followed by phosphatebuffered saline (PBS, 100 μl) and the well assembly is left for at least30 minutes to equilibrate. The well assembly is then rinsed with PBS(2×100 μl), leaving the well filled and ready for use. Into the 100 μlof PBS remaining in the well is placed a counter electrode, a connectionis made between this counter electrode and the impedance bridgemeasuring apparatus. To complete the electrical circuit, the otherconnection is made between the gold electrode and the impedance bridge.Using the impedance bridge the conduction of the membrane may ben bedetermined.

Antibody Response

The bathing PBS solution is removed from a well assembly prepared asdescribed above and replaced with a solution of monoclonalantitheophylline antibody (BioDesign Catalogue No. G45152M Clone: 8901,Lot No. 212704!, 200 nM in PBS, 100 μl). The conduction of the membraneis observed to be rapidly reduced as a consequence of the antibodyaddition. The membrane response is selective to those membranescontaining gramicidin-theophylline conjugate i.e. membranes constructedwith gramicidin derivatives bearing ligands other than theophylline(e.g. biotin) show no response to antitheophylline antibody addition.Further more, the magnitude of the response of thegramicidin-theophylline conjugate containing membranes to treatment withantitheophylline antibody is dependent on the antibody concentration andgenerates a normal titration curve with 50% response at the K_(D) of theantitheophylline-theophylline complex (FIG. 17, the ordinate shows theratio of the final initial membrane impedance at the frequency givingthe initial minimum phase angle).

Theophylline Detection

From antitheophylline-antibody-treated wells prepared as described aboveis removed 50 μl of antibody solution. A solution of theophylline in PBS(50 μl, varying concentration) is added. Above a sensitivity limit of.sup.˜ 0.1 μM, an increase in membrane conduction is observed onexposure of the antibody treated membrane to theophylline. The magnitudeof the response is related to the concentration of the theophyllinesolution, allowing construction of a dose-response curve (FIG. 18, theordinate shows the proportion of those gramicidin channels initiallygated off by antibody addition which are restored to conductivity bytheophylline treatment). The response of the membrane to theophyllinetreatment is consistent with competitive displacement ofantitheophylline antibody from the membrane-bound gramicidin-conjugatedtheophylline by free theophylline.

Synthesis

N-BOC-6-Amino Acid

To a solution of 6-aminocaproic acid (1.0 g) in 50% aqueous dioxane (12ml) was added triethylamine (2.1 ml). The mixture was stirred at roomtemperature and BOC-ON (2.71 g) was added. After stirring for 1 h at 45°C., water (15 ml) and ethyl acetate (20 ml) was added to the mixture.The aqueous phase was washed with ethyl acetate (2×20 ml), acidified topH 2 with dilute HCl and extracted with ethyl acetate. The ethyl acetatefraction was dried (Na₂ SO₄), filtered and evaporated to dryness toyield the title compound (1.64 g).

Gramacidin N-BOC-6-Aminocaproyl Ester

To a solution of gramicidin A (500 mg), N-BOC-6-aminocaproic acid (322mg) and 4-N,N-dimethylaminopyridine in dry, distilled dichoromethane (60ml) under nitrogen was added to dicyclohexylcarbodiimide (164 mg). Themixture was refluxed under nitrogen for 1 h then additionalN-BOC-6-aminocaproic acid (100 mg) and dicyclohexylcarbodiimide (110 mg)was added and the mixture was refluxed under nitrogen for another hour.The mixture was evaporated to dryness then thrice purified on a silicagel column (2×25 cm) eluted with dichloromethane/methanol/water(800:60:5) to afford an Ehrlich's reagent active fraction of gramicidinN-BOC-6-aminocaproyl ester (125 mg).

Gramicidin 6-Aminocaproyl Ester

Gramicidin N-BOC-6-aminocaproyl ester (204 mg) was twice layered withtoluene and evaporated to dryness. The residue was dried under highvacuum then treated with trifluoroacetic acid (7.5 ml) and swirled for 3min. The mixture was then evaporated to dryness. The residue was thenlayered with toluene and evaporated to dryness and dried under highvacuum. The residue was dissolved in the minimum amount ofdichloromethane/ethanol (3:1), neutralised to pH 8 with triethylamine,and precipitated with water. The precipitate was centrifuges to a pelletand the supernatant was removed by decanting. The residue was driedunder high vacuum to afford gramicidin 6-aminocaproyl ester (120 mg).

Gramicidin N-((7-Theophyllinyl)acetyl)-6-aminocaproyl Ester

A solution of gramicidin 6-aminocaproyl ester (20 mg),theophylline-7-acetic acid (3.6 mg) and2-ethoxycarbonyl-1,2-dihydoquinoline (6.2 mg) in ethanol (15 ml) wasstirred to 30 h. The solvent was then removed under reduced pressure andthe residue was chromatographed on silica gel, eluted withdichloromethane/methanol/water/acetic acid (400:40:4:1) to affordgramicidin N-((7-Theophyllinyl)acetyl)-6-aminocaproyl ester (16 mg).

What is claimed is:
 1. A membrane in which the conductance of themembrane is dependent on the presence or absence of an analyte, themembrane comprising: a first and second layer; a closely packed array ofamphiphilic molecules and a plurality of gramacidin ionophorescomprising a first and second half membrane spanning monomer, the firsthalf membrane spanning monomers being provided in the first layer andthe second half membrane spanning monomers being provided in the secondlayer, the second half membrane spanning monomers being capable oflateral diffusion within the second layer independent of the first halfmembrane spanning monomers, the first half membrane spanning monomersbeing prevented from lateral diffusion in the first layer; a firstreceptor molecule provided on at least the second half membrane spanningmonomers; and in which a proportion of the amphiphilic molecules aremembrane spanning amphiphiles which are prevented from lateral diffusionin the membrane to which are attached a second receptor molecule, thebinding of an analyte to the first and second receptor molecules causinga change in the relationship between the first half membrane spanningmonomers and the second half membrane spanning monomers such that theflow of ions across the membrane via the ionophores is allowed orprevented.
 2. A membrane as claimed in claim 1 in which the first halfmembrane spanning monomers are prevented from lateral diffusion in thefirst layer by cross-linking the monomers and the amphiphilic moleculesor by covalent attachment of the monomers to a solid surface.
 3. Amembrane as claimed in claim 1 in which the membrane spanningamphiphiles being archeobacterial lipids or tail to tail chemicallylinked bilayer amphiphiles.
 4. A membrane as claimed in claim 1 in whichthe amphiphilic molecules are membrane spanning amphiphiles, themembrane spanning amphiphiles being archeobacterial lipids or tail totail chemically linked bilayer amphiphiles.
 5. A membrane as claimed inclaim 1 in which the half membrane spanning monomers are gramicidin Amonomers.
 6. A membrane as claimed in claim 1 in which the first halfmembrane spanning monomers and the amphiphilic molecules in the firstlayer each include or are decorated with at least one moietycross-linked with at least one corresponding moiety on another of thesemolecules.
 7. A membrane as claimed in claim 1 in which the firstreceptor molecule has two receptor sites for the analyte.
 8. A membraneas claimed in claim 1 in which the first and second receptor moleculesare the same or different and are selected from the group consisting ofpolyclonal or monoclonal antibodies, antibody fragments, enzymesantigens, lectins, haptens, chelating agents and dyes.
 9. A membrane asclaimed in claim 8 in which the first and second receptor molecules areantibodies or antibody fragments.
 10. A membrane as claimed in claim 1in which the membrane is attached to a solid surface.
 11. A membrane asclaimed in claim 1 in which the second layer has a phase transitiontemperature such that cooling of the membrane below this temperatureinduces phase separation in the second layer thereby releasing anyanalyte bound to the first and/or second receptor molecules.
 12. Amembrane as claimed in claim 1 in which the first receptor molecule isattached to the second half membrane spanning monomer via a linkergroup, the linker group comprising a spacer group and one or morereactive groups, the spacer group being attached to the second halfmembrane spanning monomer and the reactive group providing attachment tothe receptor molecule.
 13. A membrane as claimed in claim 12 in whichthe spacer group is selected from the group consisting of hydrocarbons,oligomers of ethylene glycol, and oligo peptides; and the reactive groupis selected from the group consisting of N-hydrosuccinimide esters,esters for covalent coupling to amine groups on proteins, hydrazinederivatives for coupling onto oxidised sugar residues, maleiimidederivatives, biotin, streptavidin and antibodies.
 14. A membrane asclaimed in claim 1 in which the first and second receptor molecules bindto different sites on the analyte, such that binding of the first andsecond receptors to the analyte results in a change in the flow of ionsacross the membrane.
 15. A membrane as claimed in claim 1 in which thefirst and second receptor molecules are bound to different sites on theanalyte or an analog thereof, the addition of analyte effectingcompetitive binding with the first and second receptor moleculesresulting in a change in the flow of ions across the membrane.
 16. Amembrane as claimed in claim 1 in which the first receptor moleculebinds to the second receptor molecule.
 17. A membrane in which theconductance of the membrane is dependent on the presence or absence ofan analyte, the membrane comprising: a first and second layer; a closelypacked array of amphiphilic molecules and a plurality of gramacidinionophores comprising a first and second half membrane spanning monomer,the first half membrane spanning monomers being provided in the firstlayer and the second half membrane spanning monomers being provided inthe second layer, the second half membrane spanning monomers beingcapable of lateral diffusion within the second layer independent of thefirst half membrane spanning monomers, the first layer being incrystalline phase such that the first half membrane spanning monomersare prevented from lateral diffusion in the first layer; and a firstreceptor molecule provided on at least the second half membrane spanningmonomers, said first receptor molecule binding with the analyte or aportion thereof, the binding of the analyte to the first receptormolecule causing a change in the relationship between the first halfmembrane spanning monomers and the second half membrane spanningmonomers such that the flow of ions across the membrane via theionophores is allowed or prevented.
 18. A membrane as claimed in claim17 in which a proportion of the amphiphlic molecules are membranespanning amphiphiles, the membrane spanning amphiphiles beingarcheobacterial lipids or tail to tail chemically linked bilayeramphiphiles.
 19. A membrane as claimed in claim 17 in which theamphiphilic molecules are membrane spanning amphiphiles, the membranespanning amphiphiles being archeobacterial lipids or tail to tailchemically linked bilayer amphiphiles.
 20. A membrane as claimed inclaim 17 in which the half membrane spanning monomers are gramicidin Amonomers.
 21. A membrane as claimed in claim 17 in which the membraneincludes a plurality of second receptor molecules having receptor sitesremote from the first layer.
 22. A membrane as claimed in claim 21 inwhich the second receptor molecules are prevented from diffusinglaterally within the membrane.
 23. A membrane as claimed in claim 22 inwhich the second receptor molecules are attached to membrane spanningamphiphiles.
 24. A membrane as claimed in claim 17 in which the firstreceptor molecule has two receptor sites for the analyte.
 25. A membraneas claimed in claim 21 in which the first and second receptor moleculesare the same or different and are selected from the group consisting ofpolyclonal or monoclonal antibodies, antibody fragments, enzymes,antigens, lectins, haptens chelating agents and dyes.
 26. A membrane asclaimed in claim 25 in which the first and second receptor molecules areantibodies or antibody fragments.
 27. A membrane as claimed in claim 17in whch the membrane is attached to a solid surface.
 28. A membrane asclaimed in claim 21 in which the second layer has a phase transitiontemperature such that cooling of the membrane below this temperatureinduces phase separation in the second layer thereby releasing anyanalyte bound to the first and/or second receptor molecules.
 29. Amembrane as claimed in claim 17 in which the first receptor molecule isattached to the second half membrane spanning monomer via a linkergroup, the linker group comprising a spacer group and one or morereactive groups, the spacer group being attached to the second halfmembrane spanning monomer and the reactive group providing attachment tothe receptor molecule.
 30. A membrane as claimed in claim 29 in whichthe spacer group is selected from the group consisting of hydrocarbons,oligomers of ethylene glycol, and oligo peptides; and the reactive groupis selected from the group consisting of N-hydrosuccinimide esters,esters for covalent coupling to amine groups on proteins, hydrazinederivatives for coupling onto oxidised sugar residues, maleiimidederivatives, biotin, streptavidin and antibodies.
 31. A membrane asclaimed in claim 29 in which the reactive group is biotin,biotin-streptavidin or an antibody or antibody fragment directed againstthe Fc portion of an antibody.
 32. A membrane as claimed in claim 17 inwhich the first receptor molecules on separate second half membranespanning monomers bind to different sites on the analyte, such thatbinding of the first receptors to the analyte results in a change of theflow of ions across the membrane.
 33. A membrane as claimed in claim 17in which the first receptor molecules on separate second half membranespanning monomers are bound to different sites on the analyte or ananalog thereof, such that the flow of ions across the membrane isprevented, the addition of analyte effecting competitive binding withthe first receptor molecules resulting in the flow of ions across themembrane.